European Journal of Pharmacology 656 (2011) 4551

Contents lists available at ScienceDirect

European Journal of Pharmacology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Neuropharmacology and Analgesia

Geranylgeraniol and 6,7-dihydroxyvouacapan-17-oate methyl ester isolated

from Pterodon pubescens Benth.: Further investigation on the antinociceptive
mechanisms of action
Humberto M. Spindola a,b, Leila Servat a,b, Rodney A.F. Rodrigues a, Ilza M.O. Sousa a,
Joo E. Carvalho a,b, Mary A. Foglio a,b,
a
b

CPQBA, University of Campinas, P.O. Box 6171, 13083-970 Campinas, SP, Brazil
Department of Pharmacology, Anesthesiology and Therapeutics, Faculty of Dentistry, University of Campinas, P.O. Box 52, 13414-903 Piracicaba, SP, Brazil

a r t i c l e

i n f o

Article history:
Received 22 April 2010
Received in revised form 2 December 2010
Accepted 14 January 2011
Available online 4 February 2011
Keywords:
Analgesia
Pain mechanism
Natural product

a b s t r a c t
The crude alcoholic extracts obtained from Pterodon pubescens Benth. seeds are widely used in Brazilian folk
medicine as anti-inammatory, analgesic, anti-rheumatic tonics and depurative preparations. We previously
demonstrated the antinociceptive activity on writhing capsaicin, glutamate, and hot-plate tests of two
compounds isolated from P. pubescens: geranylgeraniol (C1) and 6,7-dihydroxyvouacapan-17-oate
methyl ester (C2). This work is a continuation of the previous study investigating the possible mechanisms of
action for compounds C1 and C2, and the differences between them. The present study demonstrated that
when administered intraperitoneally (i.p.): i), compounds C1 and C2 produced signicant anti-allodynic
activity during the acute phase of the Complete Freund's Adjuvant (CFA)-induced persistent pain model;
ii) compound C1 produced signicant anti-hypernociception activity in the carrageenan-induced pain model;
iii) compound C2 presented a signicant loss of activity after p-chlorophenylalanine methyl ester
hydrochloride (PCPA) [5-HT synthesis inhibitor] treatment, suggesting that the mechanisms of action could
be related to either the synthesis or release of serotonin; iv) compound C1 presented a signicant loss of
activity after ondansetron (5-HT3 receptor antagonist) treatment suggesting activity upon 5-HT3 serotonin
receptors; v) compound C1 presented a signicant loss of activity after efaroxan (mixed I1 imidazoline/
2-adrenoceptor antagonist) treatment suggesting the participation of this compound upon imidazoline I1
receptors; and vi) both compounds C1 and C2 did not appear to exert their activity via 5-HT1A, 5-HT2A,
imidazoline I2, 2-adrenoceptor, nitric oxide, GABAA, acetylcholine muscarinic, and nicotinic receptors when
evaluated in acetic acid-induced nociception.
Crown Copyright 2011 Published by Elsevier B.V. All rights reserved.

1. Introduction
Pterodon pubescens Benth. (Leguminosae) seeds are commercially
available at the Brazilian medicinal ora market and the crude alcoholic
extract of this plant is used in folk medicine in anti-inammatory,
analgesic, and anti-rheumatic preparations (Pio Correa, 1975; Lorenzi,
1998). Phytochemical studies of Pterodon genus have revealed the
presence of alkaloids, isoavones, and diterpenes. Furan diterpenes
were identied and isolated from Pterodon species (Mahjan and
Monteiro, 1973; Fascio et al., 1975; Campos et al., 1994; Arriaga et al.,

Corresponding author at: Department of Pharmacology, Anesthesiology and

Therapeutics, Faculty of Dentistry, University of Campinas, P.O. Box 52, 13414-903
Piracicaba, SP, Brazil. Tel.: +55 19 21392862; fax: +55 19 21392852.
E-mail addresses: hmspindola@fop.unicamp.br (H.M. Spindola),
leservat@hotmail.com (L. Servat), rodney@cpqba.unicamp.br (R.A.F. Rodrigues),
ilza@cpqba.unicamp.br (I.M.O. Sousa), carvalho@cpqba.unicamp.br (J.E. Carvalho),
foglioma@cpqba.unicamp.br (M.A. Foglio).

2000; Spindola et al., 2009). Our previous studies and those by other
research groups have demonstrated that furan diterpenes possessing
vouacapan skeleton are involved with the anti-inammatory, antinociceptive and antiproliferative properties of P. pubescens seed oil
(Nunan et al., 1982; Carvalho et al., 1999; Silva et al., 2004; Spindola
et al., 2009, 2010). Diterpenes 6-hydroxyvouacapan-7-17-lactone
and 6,7-dihydroxyvouacapan-17-oate methyl ester, found in
P. emarginatus and P. polygalaeorus seeds, respectively, were previously
reported to be associated with the anti-inammatory activity of these
species (Nunan et al., 1982). Different authors (Duarte et al., 1996; Silva
et al., 2004; Coelho et al., 2005; Spindola et al., 2010) have suggested a
participation of vouacapan compounds in antinociceptive and antiinammatory activity.
We have previously demonstrated the antinociceptive properties
of compounds geranylgeraniol (C1) and 6,7-dihydroxyvouacapan17-oate methyl ester (C2) isolated from P. pubescens Benth. when
evaluated on writhing, capsaicin, glutamate and hot-plate animal
experimental models (Spindola et al., 2010). In the present study, we

0014-2999/$ see front matter. Crown Copyright 2011 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2011.01.025

46

H.M. Spindola et al. / European Journal of Pharmacology 656 (2011) 4551

examined the potential anti-allodynic, anti-hypernociceptive effects,

and some of the mechanisms involved in the antinociceptive
properties (writhing test) of compounds C1 and C2. Considering
that different states of pain evoke different changes, models of pain in
integrated systems are essential to determine the potential analgesic
activity of new drugs (Suzuki and Dickenson, 2005). For this reason,
different animal models were used to better understand the
antinociceptive properties observed for the compounds.
2. Materials and Methods
2.1. Phytochemistry
2.1.1. Plant Material
Seeds were previously collected in So Paulo state (So Carlos
city), on March 2006 and identied by Prof. Dr. Jorge Yoshio
Tamashiro from IB-UNICAMP (Department of Botany) who identied
the plant species. A voucher specimen was deposited at the University
of Campinas (UEC) Herbarium, under number 1402 (Spindola et al.,
2009, 2010).
2.1.2. Compound Isolation
The compounds were isolated and identied in a previous work by
our group (Spindola et al., 2009, 2010). Briey, compounds C1 and C2
were obtained under successive column chromatography of the crude
dichloromethane extract obtained from P. pubescens seeds (Fig. 1).
2.2. Pharmacology
2.2.1. Drugs
All drugs and compounds C1 and C2 were diluted with Tween80 1%
(Sigma-Aldrich, U.S.A.) in 0.9% saline solution (NaCl diluted in
distilled water). The following substances were used: acetic acid,
Complete Freund's Adjuvant (CFA), p-chlorophenylalanine methyl
ester hydrochloride (PCPA), pindolol, ketanserin, ondansetron hydrochloride, yohimbine hydrochloride, clonidine hydrochloride, Nnitro-L-arginine (L-NAME), L-arginine hydrochloride (L-ARG), efaroxan hydrochloride, idazoxan hydrochloride, bicuculline methiodide,
atropine, mecamylamine, and carrageenan (Sigma-Aldrich, U.S.A.).
2.2.2. Animals
Male Swiss mice (2535 g) and Wistar rats (150250 g) were kept
at 25 2 C exposed to 12 h lightdark cycles (the light phase starting
at 7:00 am) and maintained in animal facilities (10 and 5 animals per
cage, respectively) with water and food ad libitum, for at least 7 days
prior to the assays. Separate groups of mice and rats were used only
once for each experiment, except on the mechanical allodynia and
hyperalgesia evaluation, where the same groups were used in each
treatment during the whole experiment. The intraperitoneal (i.p.)

Fig. 1. Chemical structures of compounds A) geranylgeraniol (C1), and B) 6,7dihydroxyvouacapan-17-oate methyl ester (C2).

route was used for all experiments, based on a previous report

(Spindola et al., 2010). The studies were carried out in accordance
with the current guidelines for veterinary care of laboratory animals
(Voipio et al., 2008) and were performed under the consent and
surveillance of Unicamp's Institute of Biology Ethics Committee for
Animal Research (1076-1).
2.2.3. Mechanical Allodynia Induced by Complete Freund's Adjuvant
(CFA)
The procedures were developed and standardized in our laboratory based on the method previously described (Villeti et al., 2003)
with changes in protocol and data analysis. Different groups of rats
(n = 5) were used during the whole experiment and inammation
was induced with a solution of CFA (1 mg/ml of heat killed
Mycobacterium tuberculosis in 85% parafn oil and 15% mannide
monooleate) injected (0.1 ml) into the plantar surface of the right
hind paw. The left hind paw received the same volume of saline
solution (NaCl 0.9% diluted in distilled water) in order to equalize the
sensibility of the animals caused by the injection. Mechanical
allodynia was assessed using the Dynamic Plantar Aesthesiometer
apparatus (Ugo Basile, mod 37450, Italy) which consisted of an
elevated wire mesh platform to allow access to the ventral surface of
the hind paws. A steel rod (diameter 0.5 mm) was pushed against the
hind paw with ascending force (touch stimulator). The force ranged
from 0 to 35 g over a 20-s period. When the animal withdrew the hind
paw, the mechanical stimulus was automatically stopped, and the
force applied by the animal to withdraw the paw was recorded to the
nearest 0.1 g. An allodynia score was determined after four consecutive measurements using the touch stimulator sequentially on the
left and right hind paw and calculated considering the formula below
determined by the authors:
Left hind paw value = Right hind paw value = Allodynia score:
The basal score was measured before CFA injection on day 0, and
the animals considered for testing were those with a mean value
nearest to 1 (demonstrating no signicant difference between both
paw stimuli). After CFA injection, measurements were carried out
considering three different phases, as follows: 4 h on day 0 (acute
pain); 24 h on day 1 (sub-acute pain) and on day 14 (chronic pain).
Vehicle (10 ml/kg) or compounds C1 and C2 were administered
(30 mg/kg, i.p.) 30 min prior to touch stimulation, in order to evaluate
the possible anti-allodynic activity observed for each phase. A positive
control was not employed, as the aim of this test was to evaluate the
activity of compounds using the same doses to those used in the tests
described below for evaluating potential antinociceptive mechanisms.
2.2.4. Mechanical Hyperalgesia Induced by Carrageenan
The procedures used for this study were similar to those described
previously (Randall and Selitto, 1957) with some changes in the
protocol and data analysis. Different groups of rats (n = 6), used
during the whole experiment, were submitted to pressure stimulus (0
to 500 g) on the right hind paw using an Analgesy-meter (Ugo Basile,
mod 37215/372116, Italy) prior to carrageenan injection, in order to
determine the basal value. The hypernociceptive response was
considered when animals vocalized or withdrew the paw from the
equipment, demonstrating pain. After this rst measurement, animals
received a carrageenan (0.1 ml) intraplantar (i.pl.) injection (2.5% in
saline) into the right hind paw surface. After 2:30 h, they were
submitted to pressure again, to evaluate whether the hypernociceptive state was reached (40% decrease). Animals were treated with
vehicle (negative control), indomethacin (30 mg/kg, i.p. positive
control) and compounds C1 and C2 (30 mg/kg, i.p.), and the
mechanical hyperalgesia was evaluated after 30 min, 1 h, 2 h and
3 h. Compound doses were dened according to previous experiments (Spindola et al., 2010). The value demonstrating mechanical

H.M. Spindola et al. / European Journal of Pharmacology 656 (2011) 4551

47

hyperalgesia was obtained after each measurement, and the results

were shown as decreased percentage compared to the pressure
tolerated in the basal (100%) value for each group.
2.2.5. Abdominal Constriction Induced by Acetic Acid (Writhing Test)
Abdominal constrictions were induced according to procedures
previously described (Spindola et al., 2010), and resulted in abdominal
muscle contraction and concomitant stretching of the hind limbs, in
response to an i.p. injection of 0.8% acetic acid (10 ml/kg) at the time of
the test. The number of abdominal constrictions during 15 min was
indicative of nociception. For these evaluations, a number of male Swiss
mice (6 to 8) were used.
2.2.5.1. Investigation of the Antinociceptive Mechanisms of Compounds
C1 and C2. To address some of the antinociceptive mechanisms of C1
and C2, male Swiss mice were pre-treated with different receptor
antagonists in the writhing test. Response thresholds were measured
30 min after the second injection. The doses of each receptor
antagonist were selected based on other experiments reported in
the literature (Santos et al., 2005; Dalb et al., 2006; Yue et al., 2007)
and on our preliminary experiments (Spindola et al., 2010).
2.2.5.1.1. Involvement of Serotonergic System. To explore the
possible participation of the serotonergic system in the antinociceptive action of compounds C1 and C2, mice were pre-treated with
either PCPA (a 5-HT synthesis inhibitor, once a day, during 4
consecutive days before testing, 100 mg/kg, i.p.), pindolol (a 5-HT1A
receptor antagonist, 1 mg/kg, i.p.), ketanserin (a 5-HT2A receptor
antagonist, 0.3 mg/kg, i.p.), ondansetron (a 5-HT3 receptor antagonist,
0.2 mg/kg, i.p.), or vehicle (10 ml/kg), and 20 min after they then
received compounds C1 and C2 (30 mg/kg, i.p.) or vehicle, 30 min
prior to acetic acid injection.
2.2.5.1.2. Involvement of Imidazoline System. Aiming to evaluate the
involvement of imidazoline receptors in the antinociceptive action of
compounds C1 and C2, the mice were pre-treated with efaroxan (a mixed
I1 imidazoline/2-adrenoceptor antagonist, 2 mg/kg, i.p.), idazoxan
(a mixed I2 imidazoline/2-adrenoceptor antagonist, 10 mg/kg, i.p.) or
vehicle (10 ml/kg, i.p.), 20 min before administration of compounds C1
and C2 (30 mg/kg), clonidine (an 2-adrenoceptor agonist, 0.1 mg/kg,
i.p.), or vehicle (10 ml/kg, i.p.), and after 30 min they received the
injection of acetic acid.

Fig. 2. Graph demonstrating decrease on allodynia score related to the touch

stimulation exerted on the surface of the left and right hind paws caused by injection
of saline solution and CFA (0.1 ml) respectively, producing persistent pain sensitization
in rats treated with compounds geranylgeraniol (C1) and 6,7-dihydroxyvouacapan17-oate methyl ester (C2) (30 mg/kg, i.p.) compared to the control group (vehicle,
10 ml/kg). Results expressed as reduction score means S.E.M. of 5 animals for
experimental groups (*P 0.05).

3.3. Evaluation of Some Potential Antinociceptive Mechanisms

of Geranylgeraniol (C1) and 6,7-dihydroxyvouacapan-17-oate
Methyl Ester (C2)
3.3.1. Participation of Serotonergic System
The results presented in Figs. 47 showed that compounds C1 and
C2 (30 mg/kg, i.p.) produced a signicant antinociception reducing
writhes in mice, compared to the control group (vehicle only). They
also showed that:
The pre-treatment (once a day) during four consecutive days, with
5-HT synthesis inhibitor PCPA (100 mg/kg, i.p.) signicantly
reversed the antinociception caused by compound C2, however
not by C1 (Fig. 4);

2.2.6. Statistical Analysis

All results were submitted to one way analysis of variance
(ANOVA) with repeated measurements, P 0.05 was considered the
critical level for evaluating signicant difference between the control
and treated groups, followed by Duncan's test, using StatSoft
software. Graphs were designed using the Origin software.
3. Results
3.1. Mechanical Allodynia
The results presented in Fig. 2 show that compounds C1 and C2
(30 mg/kg, i.p.) produced signicant anti-allodynic activity reducing
the allodynic score compared to the control (vehicle) during the acute
phase (4 h post CFA) of the test. The compounds exerted no activity
during the sub-chronic and chronic phases.
3.2. Mechanical Hyperalgesia
The results presented in Fig. 3 showed that control indomethacin
(30 mg/kg, i.p.) and compound C1 (30 mg/kg, i.p.) produced significant anti-hypernociceptive effect increasing the percentage of
stimulus compared to the control group (vehicle), however C2 did
not.

Fig. 3. Graph demonstrating results of vocalization or paw withdrawal related to

pressure exerted in the right hind paw in response to the hypernociception caused by
injection of carrageenan (2.5%, 0.1 ml) in rats treated with indomethacin, compounds
geranylgeraniol (C1) and 6,7-dihydroxyvouacapan-17-oate methyl ester (C2)
(30 mg/kg, i.p.) compared to the control group (vehicle). Results expressed as
reduction percentage means S.E.M. of 5 animals for experimental groups compared
to the basal values (considered 100%) (*P 0.05; **P 0.01; ***P 0.001).

48

H.M. Spindola et al. / European Journal of Pharmacology 656 (2011) 4551

Fig. 4. Graph demonstrating effect of pre-treatment of animals with PCPA (100 mg/kg, 4
consecutive days, i.p.) on the antinociceptive proles of geranylgeraniol (C1) and
6,7-dihydroxyvouacapan-17-oate methyl ester (C2) (30 mg/kg, i.p.) against the
acetic acid-induced writhing in mice. Each column represents the mean of 6 to
8 animals and the error bar indicates the S.E.M. P 0.05, P 0.01, P 0.001
compared with corresponding control values (injected with vehicle alone); #P 0.05
comparing to the respective agonist group (reversing effect of compounds C1 or C2).

Pre-treatment with 5-HT1A receptor antagonist pindolol (1 mg/kg,

i.p.), did not reverse the antinociception caused by both compounds C1 and C2 (Fig. 5);
Pre-treatment with 5-HT 2A receptor antagonist ketanserin
(0.3 mg/kg, i.p.), did not reverse the antinociception caused by
both compounds C1 and C2 (Fig. 6);
The pre-treatment with 5-HT3 receptor antagonist ondansetron
(0.2 mg/kg, i.p.) signicantly reversed the antinociception caused
by compound C1, however not by C2 (Fig. 7).
3.3.2. Participation of Imidazoline System
The results presented in Fig. 8 demonstrated that compounds C1,
C2 (30 mg/kg, i.p.), and the 2-adrenoceptor agonist clonidine
(0.1 mg/kg, i.p.) produced signicant antinociception which was

Fig. 5. Graph demonstrating effect of pre-treatment of animals with pindolol (1 mg/kg, i.p.)
on the antinociceptive proles of geranylgeraniol (C1) and 6,7-dihydroxyvouacapan17-oate methyl ester (C2) (30 mg/kg, i.p.) against the acetic acid-induced writhing in
mice. Each column represents the mean of 6 to 8 animals and the error bar indicates the
S.E.M. P 0.001 compared with corresponding control values (injected with vehicle
alone).

Fig. 6. Graph demonstrating effect of pre-treatment of animals with ketanserin

(0.3 mg/kg, i.p.) on the antinociceptive proles of geranylgeraniol (C1) and 6,7dihydroxyvouacapan-17-oate methyl ester (C2) (30 mg/kg, i.p.) against the acetic
acid-induced writhing in mice. Each column represents the mean of 6 to 8 animals and
the error bar indicates the S.E.M. P 0.001 compared with corresponding control
values (injected with vehicle alone).

conrmed by the reduction of writhes in mice, compared to the

control group (vehicle only). The pre-treatment with the mixed I1
imidazoline/2-adrenoceptor antagonist efaroxan (2 mg/kg, i.p.)
signicantly reversed the antinociception caused by clonidine (2adrenoceptor agonist, 0.1 mg/kg, i.p.) and compound C1 (30 mg/kg, i.
p.), however not by C2. Whereas, the pre-treatment with the mixture
I2 imidazoline/2-adrenoceptor antagonist idazoxan (10 mg/kg, i.p.),
reversed only the antinociception caused by clonidine.
3.3.3. Participation of Other Systems
The results presented in Online supplement support the information that compounds C1 and C2 do not appear to exert activity via 2adrenoceptor, nitric oxide (NO), inhibitory GABAA, acetylcholine
muscarinic, and nicotinic receptors when evaluated in the acetic
acid-induced nociception.

Fig. 7. Graph demonstrating effect of pre-treatment of animals with ondansetron

(0.2 mg/kg, i.p.) on the antinociceptive proles of geranylgeraniol (C1) and 6,7dihydroxyvouacapan-17-oate methyl ester (C2) (30 mg/kg, i.p.) against the acetic
acid-induced writhing in mice. Each column represents the mean of 6 to 8 animals and
the error bar indicates the S.E.M. P 0.05, P 0.01, compared with corresponding
control values (injected with vehicle alone); #P 0.05 comparing to the respective
agonist group (reversing effect of compounds C1 or C2).

H.M. Spindola et al. / European Journal of Pharmacology 656 (2011) 4551

Fig. 8. Graph demonstrating effect of pre-treatment of animals with efaroxan (2 mg/kg,

i.p.) and idazoxan (10 mg/kg, i.p.) on the antinociceptive proles of geranylgeraniol
(C1) and 6,7-dihydroxyvouacapan-17-oate methyl ester (C2) (30 mg/kg, i.p.)
against the acetic acid-induced writhing in mice. Each column represents the mean of 6
to 8 animals and the error bar indicates the S.E.M. P 0.05, P 0.001 compared with
corresponding control values (injected with vehicle alone); #P 0.01, ##P 0.001
comparing to the respective agonist group (reversing effect of compounds C1, C2 or
clonidine 0.1 mg/kg).

4. Discussion
We recently reported a few antinociceptive properties of compounds geranylgeraniol (C1) and 6,7-dihydroxyvouacapan-17oate methyl ester (C2) isolated from P. pubescens Benth. (Fig. 1)
suggesting the exclusion of opioid receptors when evaluated through
the hot-plate test (Spindola et al., 2010). In the present study, we
extend the previous data, investigating the potential anti-allodynic
and anti-hypernociceptive effects, and some antinociceptive mechanisms for C1 and C2 using the writhing test, in order to understand
better their clinical potential use for pain relief. The most relevant
ndings were that given intraperitoneally (i.p.): i) compounds C1 and
C2 produced signicant anti-allodynic effect during the acute phase of
CFA-induced persistent pain model; ii) compound C1 produced
signicant anti-hypernociceptive effect upon the carrageenan-induced pain model; iii) the antinociceptive action of compounds C1
and C2 during the writhing test could be related to serotonergic and
imidazoline systems.
Pain is normally a transitory unpleasant sensation subsequent to a
noxious or potentially injurious stimulus generated in somatic and
visceral tissues. Unlike acute pain, inammatory and neuropathic
pains are often persistent, chronic states. Patients suffering from
chronic pain often experience hypersensitivity to mechanical, thermal
and chemical stimulation in the form of hyperalgesia (aggravated pain
response to normally painful stimuli) and/or allodynia (pain response
to innocuous stimuli) (Levine and Alessandri-Haber, 2007). Our
previous work demonstrated the effectiveness of compounds C1 and
C2 on the writhing, hot plate, capsaicin and glutamate tests,
suggesting their activities related to vanilloid and/or glutamatergic
receptors (Spindola et al., 2010). A growing amount of experimental
data indicates that these receptors are involved in the mechanism of
pain and inammation, and their antagonists might exhibit clinically
potential relevance in the management of pathological pain states,
such as chronic pain (Woolf and Thompson, 1991). Considering the
diverse etiologies and the variety of molecular mechanisms underlying pain hypersensitivity, the approach of targeting ion channels in
primary afferent nociceptive neurons that can contribute to the
detection of physical stimuli, may be an effective approach for
developing more successful therapies for clinical pain syndromes

49

(Levine and Alessandri-Haber, 2007). To assess the effects of a 30 mg/

kg (dened by previous data based on compounds ED50) doses of
compounds C1 and C2 given intraperitoneally (i.p.) in persistent
models of pain, we analyzed the mechanical anti-allodynic and antihypernociceptive actions induced by intraplantar injections of
Complete Freund's Adjuvant (CFA) and carrageenan, respectively.
Both models of persistent pain used in the present study produce
central sensitization in response to the release of several proinammatory mediators, which increase the sensitivity of peripheral
and central sensory pathways (Basbaum, 1999; Minami et al., 2006).
Persistent pain caused by CFA intraplantar injection involves
central sensitization due to the release of multiple inammatory and
pain mediators that account for sensitivity increase of both peripheral
sensory afferents at the injury site, and in the central nervous system
(Samad et al., 2001). The arrival of sensorial information from
nociceptors into the dorsal horn considerably alters the level of
activity within the cord as both excitatory and inhibitory systems can
impinge upon spinal neuronal activity. This forms the basis of central
hypersensitivity which results in increased responsiveness of dorsal
horn neurons, often observed in persistent inammatory and
neuropathic states of pain (Suzuki and Dickenson, 2005). Our results
showed that the mechanical anti-allodynic effects of compounds C1
and C2 (30 mg/kg, i.p.) were observed on the acute phase of the test
(4 h post CFA). These effects were not observed on the sub-acute and
chronic phases (Fig. 2).
Consequently, the anti-hypernociceptive effects of both C1 and C2
were evaluated in the carrageenan-induced hyperalgesia, consisting
in a neutrophil-mediated acute inammatory response that produces
hind paw swelling, erythematic and localized hyperthermia induced
by an injection of carrageenan into the rats paw (Cunha et al., 2005).
This inammatory method has become a widely used model for
studying acute inammation (Winter et al., 1962). In this model,
carrageenan evokes a very inammatory and nociceptive response
characteristic, which is mediated by different groups of endogenous
substances that stimulate chemosensitive nociceptors, thus playing a
major role in the development of inammatory pain (Posadas et al.,
2004). Our results also showed that compound C1 was effective in this
assay (Fig. 3) reducing mechanical hyperalgesia of rats, presenting an
important difference compared to C2 which did not produce the same
effect.
Moreover, in the present study we attempted to characterize
further some of the mechanisms by which compounds C1 and C2 may
exert their antinociceptive action in a chemical model of nociception
in mice (writhing test), evaluating a few specic receptors in different
pain systems. We assessed the following pathways to evaluate some
receptors involved in the antinociceptive mechanisms: serotonergic,
2-adrenergic, imidazoline, nitric oxide, inhibitory GABAA, neuronal
nicotinic acetylcholine, and muscarinic acetylcholine systems. However, only the serotonergic and imidazoline systems seemed to be
involved, as described below.
In CNS, serotonin (5-HT) neurons are involved in nociceptive
transmission as well as in pain inhibition induced by opioid agonists.
Serotonin has also been observed to produce an inhibitory effect on
pain transmission in the spinal cord and brain (Yaksh and Wilson,
1079). This inhibitory effect could be mediated by 5-HT receptors
inuencing the descending inhibitory system (Sawynok and Reid,
1996). Our ndings demonstrated that compounds C1 and C2 may
exert their antinociceptive properties, at least in part, via serotonergic
system. This assertion is supported by the demonstration that
depletion of endogenous serotonin with the tryptophan hydroxylase
inhibitor p-chlorophenylalanine methyl ester hydrochloride (PCPA)
at a dose known to decrease the cortical content of serotonin and to
signicantly reverse the morphine antinociception, largely antagonized the antinociception of C2 (though not C1), suggesting that the
action of the compound could be due to 5-HT synthesis or release
inuencing (Fig. 4). In addition, specic 5-HT receptors 5-HT1, 5-HT2A,

50

H.M. Spindola et al. / European Journal of Pharmacology 656 (2011) 4551

and 5-HT3 were investigated in the same visceral model, throughout

the pretreatment with the respective antagonists: pindolol, ketanserin
and ondansetron.
The selective antagonist of 5-HT3 receptors ondansetron, consistently reversed the antinociception provoked by C1 administration
when using the writhing test (Fig. 7). Contrarily, neither compound
seemed to exert antinociceptive activity upon 5-HT1A or 5-HT2A,
because the pretreatment with the specic antagonists, pindolol and
ketanserin (respectively), did not block the antinociceptive action of
both compounds on the same model (Figs. 5 and 6). Considering that
supraspinal serotonergic inputs maintain the facilitatory inuences of
the spinal cord following injury (Wang et al., 2002), we speculated
that C1 could act directly upon the 5-HT3 receptor, and that C2 could
act by increasing or decreasing the serotonin amount, inuencing the
descending inhibitory pathway.
The imidazoline I1-receptor has the best understood physiologic
actions among the reported imidazoline receptors, and has been
implicated in hypotension produced by clonidine and other imidazolines when injected into the rostral ventrolateral medulla (RVLM) or
administered peripherally (Ernsberger et al., 1997; Head et al., 1997).
The I1-receptor has been further implicated in the modulation of
gastric acid secretion, analgesia and modulation of electrolyte kidney
secretion (Smyth et al., 1995). Another interesting nding of the
present work is that the results herein presented provided consistent
evidence supporting imidazoline I1 receptor involvement in the
antinociception caused by C1, evident due to the fact that efaroxan,
at a similar dose to that known to prevent antinociception induced by
the imidazoline I1 receptor agonist moxonidine, consistently attenuated both clonidine and C1 induced antinociception during the
writhing test (Fig. 8) (Shannon and Lutz, 2000). Contrarily, imidazoline I2 receptors do not appear to be involved in the antinociception of
C1 and C2, as the pretreatment using the preferential I2 imidazoline
receptor antagonist idazoxan, largely failed to prevent the antinociception of both compounds. The present data allowed us to verify that
the antinociception caused by C1 is probably, at least in part, linked to
an interaction with imidazoline I1 receptors.
In summary, the data of this paper demonstrated important
differences in the antinociceptive effects observed for compounds
geranylgeraniol (C1) and 6,7-dihydroxyvouacapan-17-oate
methyl ester (C2) obtained from P. pubescens Benth., being active
against several pain states and extending our previous results which
suggested a possible synergistic activity between them. When
evaluated on the proposed experimental models of pain, compound
C1 demonstrated effect against inammatory and neurogenic pain
states possibly related to glutamate, capsaicin, serotonergic (5-HT3),
and imidazoline (I1) pathways. Whereas, compound C2 produced
signicant antinociceptive effects, probably acting on glutamate and
capsaicin receptors (Spindola et al., 2010), and inuencing the
serotonergic system (inhibitory pathways), demonstrating the suggested synergistic activity among these compounds and corroborating
the analgesic efcacy of extracts and compounds obtained from
P. pubescens Benth. species.
5. Conclusion
The present study demonstrated that: 1) compounds C1 and C2
produced signicant anti-allodynic activity during the acute phase of
the CFA-induced persistent pain model; 2) compound C1 produced
signicant anti-hypernociception activity upon the carrageenan-induced pain model; 3) compound C2 signicantly lost activity after PCPA
treatment suggesting that 6,7-dihydroxyvouacapan-17-oate methyl ester mechanisms could be related either to the synthesis or release of
serotonin; 4) compound C1 signicantly lost activity after ondansetron
treatment suggesting participation of geranylgeraniol upon 5-TH3
serotonin receptors; 5) compound C1 signicantly lost activity after
efaroxan treatment suggesting participation of this compound upon

imidazoline I1 receptors; and 6) both compounds C1 and C2 do not

appear to exert activity via 5-HT1A, 5-HT2A, 2-adrenoceptor, imidazoline I2, NO, GABAA, acetylcholine muscarinic, and nicotinic receptors
when evaluated in the acetic acid-induced nociception (Supplementary
information).
Acknowledgements
The authors thank CAPES, FAPESP and CNPq for nancial support
and research fellowships.
Appendix A. Supplementary data
Supplementary data to this article can be found online at doi:10.
1016/j.ejphar.2011.01.025.
References
Arriaga, A.M., Gomes, G.A., Braz-Filho, R., 2000. Constituents of Bowdichia virgilioides.
Fitoterapia 71 (2), 211212.
Basbaum, A.I., 1999. Spinal mechanisms of acute and persistent pain. Reg. Anesth. Pain
Med. 24, 5967.
Campos, A.M., Silveira, E.R., Braz-Filho, R., Teixeira, T.C., 1994. Diterpenoids from
Pterodon polygalaeorus. Phytochemistry 36 (2), 403406.
Carvalho, J.C.T., Serti, J.A.A., Barbosa, M.V.J., Patrcio, K.C.M., Caputo, L.R.G., Sarti, S.J.,
Ferreira, L.P., Bastos, J.K., 1999. Anti-inammatory activity of the crude extract form
the fruits of Pterodon emarginatus Vog. J. Ethnopharmacol. 64, 127133.
Coelho, L.P., Reis, P.A., Castro, F.L., Gayer, C.R.M., Lopes, C.S., Silva, M.C.C., Sabino, K.C.C.,
Todeschini, A.R., Coelho, M.G.P., 2005. Antinociceptive properties of ethanolic
extract and fractions of Pterodon pubescens Benth. seeds. J. Ethnofarmacol. 98,
109116.
Cunha, M.T., Verri Jr., W.A., Silva, J.S., Poole, S., Cunha, F.Q., Ferreira, S.H., 2005. A cascade
of cytokines mediates mechanical inammatory hypernociception in mice. Proc.
Natl Acad. Sci. 102, 17551760.
Dalb, S., Jurgensen, S., Horst, H., Soethe, D.N., Santos, A.R.S., Pizzolatti, M.G., Ribeiro-doValle, R.M., 2006. Analysis of the antinociceptive affect of the proanthocyanidinrich fraction obtained from Croton celtidifolius barks: evidence for a role of the
dopaminergic system. Pharmacol. Biochem. Behav. 85, 317323.
Duarte, I.D.G., Alves, D.L.F., Veloso, D.P., Nakamura-Crag, M., 1996. Evidence of the
involvement of biogenic amines in the antinociceptive effects of vouacapan
extracted from Pterodon polygalaeorus Benth. J. Ethnopharmacol. 55, 1318.
Ernsberger, P., Friedman, J.E., Koletsky, R.J., 1997. The I1-imidazoline receptor: from
binding site to therapeutic target in cardiovascular disease. J. Hypertens. 15, 1923.
Fascio, M., Mors, W.B., Gilbert, B., Mahajan, J.R., Monteiro, M.B., Dos Santos Filho, D.,
Vichnewski, W., 1975. Diterpenoid furans from Pterodon species. Phytochemistry
15, 201203.
Head, G.A., Burke, S.L., Chan, C.K., 1997. Central imidazoline receptors and centrally
acting anti-hypertensive agents. Clin. Exp. Hypertens. 19, 591605.
Levine, J.D., Alessandri-Haber, N., 2007. TRP channels: targets for the relief of pain.
Biochim. Biophys. Acta 1772 (8), 9891003.
Lorenzi, H., 1998. rvores Brasileiras. Man. Ident. Cult. Plant. Arb. Nat. Bras. 1, 227.
Mahjan, J.R., Monteiro, M.B., 1973. New diterpenoids from Pterodon emarginatus.
J. Chem. Soc. Perkin Trans. 1, 520525.
Minami, M., Katayama, T., Satoh, M., 2006. Brain cytokines and chemokines: roles in
ischemic injury and pain. J. Pharmacol. Sci. 100, 461470.
Nunan, E.A., Carvalho, M.G., Piloveloso, D., 1982. Furane diterpenes with anti-inammatory
and pro-inammatory activity. Braz. J. Med. Biol. Res. 15 (6), 450.
Pio Correa, M., 1975. Dicionrio das plantas teis do Brasil e das exticas cultivadas.
Inst. Bras. Des. Flor. 1, 153.
Posadas, I., Bucci, M., Roviezzo, F., Rossi, A., Parente, L., Sautebin, L., Cirino, G., 2004.
Carrageenan-induced mouse paw oedema is biphasic, age-weight dependent and
displays differential nitric oxide cyclooxygenase-2 expression. Br. J. Pharmacol.
142, 331338.
Randall, R.O., Selitto, J.J., 1957. A method for measurement of analgesic activity on
inamed tissue. Arch. Int. Pharmacodyun. 111, 409419.
Samad, T.A., Moore, K.A., Sapirstein, A., Billet, S., Allchorne, A., Poole, S., Bonventre, J.V.,
Woolf, C.J., 2001. Interleukin-1b-mediated induction of Cox-2 in the CNS contributes
to inammatory pain hypersensitivity. Nature 410, 471475.
Santos, A.R.S., Gadotti, V.M., Oliveira, G.L., Tibola, D., Paszuck, A.P., Neto, A., Spindola, H.M.,
Souza, M.M., Rodrigues, A.L.S., Calixto, J.B., 2005. Mechanisms involved in the
antinociception caused by agmatine in mice. Neuropharmacology 48, 10211034.
Sawynok, J., Reid, A., 1996. Interactions of descending serotonergic systems with other
neurotransmitters in the modulation of nociception. Behav. Brain Res. 73, 6368.
Shannon, H.E., Lutz, E.A., 2000. Effects of the I(1) imidazoline/alpha(2)-adrenergic
receptor agonist moxonidine in comparison with clonidine in the formalin test in
rats. Pain 85, 161167.
Silva, M.C.C., Gayer, C.R.M., Lopes, C.S., Calixto, N.O., Reis, P.A., Passeas, C.P.B., Paes, M.C.,
Dalmau, S.R., Sabino, K.C.C., Todeschini, A.R., Coelho, M.G.P., 2004. Acute and topic
anti-edematogenic fractions isolated from the seeds of Pterodon pubescens. Pharm.
Pharmacol. 55, 135141.

H.M. Spindola et al. / European Journal of Pharmacology 656 (2011) 4551

Smyth, D.D., Darkwa, F.K., Penner, S.B., 1995. Imidazoline receptors and sodium
excretion in the rat kidney. Ann. N.Y. Acad. Sci. 763, 340352.
Spindola, H.M., Carvalho, J.E., Ruiz, A.L.T.G., Rodrigues, A.F.R., Denny, C., Sousa, I.M.O.,
Tamashiro, J.Y., Foglio, M.A., 2009. Furanoditerpenes from Pterodon pubescens
Benth with selective in vitro anticancer activity for prostate cell line. J. Braz. Chem.
Soc. 20 (3), 569575.
Spindola, H.M., Servat, L., Denny, C., Rodrigues, R.A.F., Eberlin, M.N., Cabral, E., Sousa, I.M.O.,
Carvalho, J.E., Tamashiro, J.Y., Foglio, M.A., 2010. Antinociceptive effect of geranylgeraniol and 6,7-dihydroxyvouacapan-17-oate methyl ester isolated from Pterodon
pubescens Benth. BMC Pharmacol. 10 (1), 110.
Suzuki, R., Dickenson, A., 2005. Spinal and supraspinal contributions to central
sensitization in peripheral neuropathy. Neurosignals 14, 175181.
Villeti, G., Bergamarchi, M., Bassani, F., Bolzoni, P.T., Maiorino, M., Pietra, C., Rondelli, I.,
Chamiot-Clark, P., Simonato, M., Barbieri, M., 2003. Antinociceptive activity of the
N-methyl-D-aspartate receptor antagonist N-(2-indanyl)-glycinamide hydrochloride (CHF3381) in experimental models of inammatory and neurophatic pain.
J. Pharmacol. Exp. Ther. 306, 804814.

51

Voipio, H.M., Baneux, P., Gomez de Segura, I.A., Hau, J., Wolfensohn, S., 2008. Guidelines
for the veterinary care of laboratory animals: report of the FELASA/ECLAM/ESLAV
Joint Working Group on Veterinary Care. Lab. Anim. 42 (1), 111.
Wang, H., Sun, H., Della Penna, K., Benz, R., Xu, J., Gerhold, D., Holder, D., Koblan, K., 2002.
Chronic neuropathic pain is accompanied by global changes in gene expression and
shares pathobiology with neurodegenerative diseases. Neuroscience 114, 529546.
Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenan-induced edemas in hind paw of the
rat as an assay for anti-inammatory drugs. Proc. Soc. Exp. Biol. Med. 111, 544547.
Woolf, C.J., Thompson, S.W.N., 1991. The induction and maintenance of central
sensitization is dependent on N-methyl-D-aspartic acid receptor activation:
implications for the treatment of post-injury pain hypersensitivity states. Pain
44, 293299.
Yaksh, T.L., Wilson, P.R., 1979. A spinal serotonin terminal system mediates antinociception.
J. Pharmacol. Exp. Ther. 208, 446453.
Yue, C.Q., Ye, J., Li, C.L., Li, R.T., Sun, Q., 2007. Antinociceptive effects of the novel
spirocyclopiperazinium salt compound LXM-10 in mice. Pharmacol. Biochem.
Behav. 86, 643650.